Identification of Free-B-Ring flavonoids as potent COX-2 inhibitors

ABSTRACT

The present invention provides a novel method for inhibiting the cyclooxygenase enzyme COX-2. The method is comprised of administering a composition containing a Free-B-Ring flavonoid or a composition containing a mixture of Free-B-Ring flavonoids to a host in need thereof. The present also includes novel methods for the prevention and treatment of COX-2 mediated diseases and conditions. The method for preventing and treating COX-2 mediated diseases and conditions is comprised of administering to a host in need thereof an effective amount of a composition containing a Free-B-Ring flavonoid or a composition containing a mixture of Free-B-Ring flavonoids and a pharmaceutically acceptable carrier.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/469,275, filed Aug. 27, 2003, which application is a 35 U.S.C.§371 national phase application of PCT/US03/06098 (WO 03/074065), filedon Feb. 28, 2003, which is a continuation of U.S. application Ser. No.10/091,362, filed Mar. 1, 2002 each of which is entitled “Identificationof Free-B-Ring Flavonoids as Potent COX-2 Inhibitors,”. Each of theseapplications is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to a method for the prevention andtreatment of COX-2 mediated diseases and conditions. Specifically, thepresent invention relates to a method for the prevention and treatmentof COX-2 mediated diseases and conditions by administration of compoundsreferred to herein as Free-B-Ring flavonoids. Included in this inventionis an improved method to generate standardized Free-B-Ring flavonoidextracts from plant sources.

BACKGROUND OF THE INVENTION

The liberation and metabolism of arachidonic acid (AA) from the cellmembrane, results in the generation of pro-inflammatory metabolites byseveral different pathways. Arguably, the two most important pathways toinflammation are mediated by the enzymes 5-lipoxygenase (5-LO) andcyclooxygenase (COX). These are parallel pathways that result in thegeneration of leukotrienes and prostaglandins, respectively, which playimportant roles in the initiation and progression of the inflammatoryresponse. These vasoactive compounds are chemotaxins, which both promoteinfiltration of inflammatory cells into tissues and serve to prolong theinflammatory response. Consequently, the enzymes responsible forgenerating these mediators of inflammation have become the targets formany new anti-inflammatory drugs.

Inhibition of the enzyme cyclooxygenase (COX) is the mechanism of actionattributed to most nonsteroidal anti-inflammatory drugs (NSAIDS). Thereare two distinct isoforms of the COX enzyme (COX-1 and COX-2) that shareapproximately 60% sequence homology, but differ in expression profilesand function. COX-1 is a constitutive form of the enzyme that has beenlinked to the production of physiologically important prostaglandins,which help regulate normal physiological functions, such as plateletaggregation, protection of cell function in the stomach and maintenanceof normal kidney function. (Dannhardt and Kiefer (2001) Eur. J. Med.Chem. 36:109-26). The second isoform, COX-2, is a form of the enzymethat is inducible by pro-inflammatory cytokines, such as interleukin-1β(IL-1β) and other growth factors. (Herschmann (1994) Cancer MetastasisRev. 134:241-56; Xie et al. (1992) Drugs Dev. Res. 25:249-65). Thisisoform catalyzes the production of prostaglandin E2 (PGE2) fromarachidonic acid (AA). Inhibition of COX-2 is responsible for theanti-inflammatory activities of conventional NSAIDs.

Because the mechanism of action of COX-2 inhibitors overlaps with thatof most conventional NSAID's, COX-2 inhibitors are used to treat many ofthe same symptoms, including pain and swelling associated withinflammation in transient conditions and chronic diseases in whichinflammation plays a critical role. Transient conditions includetreatment of inflammation associated with minor abrasions, sunburn orcontact dermatitis, as well as, the relief of pain associated withtension and migraine headaches and menstrual cramps. Applications tochronic conditions include arthritic diseases, such as rheumatoidarthritis and osteoarthritis. Although, rheumatoid arthritis is largelyan autoimmune disease and osteoarthritis is caused by the degradation ofcartilage in joints, reducing the inflammation associated with eachprovides a significant increase in the quality of life for thosesuffering from these diseases. (Wienberg (2001) Immunol. Res. 22:319-41;Wollhiem (2000) Curr. Opin. Rheum. 13:193-201). In addition torheumatoid arthritis, inflammation is a component of rheumatic diseasesin general. Therefore, the use of COX inhibitors has been expanded toinclude diseases, such as systemic lupus erythromatosus (SLE) (Goebel etal. (1999) Chem. Res. Tox. 12:488-500; Patrono et al. (1985) J. Clin.Invest. 76:1011-1018), as well as, rheumatic skin conditions, such asscleroderma. COX inhibitors are also used for the relief of inflammatoryskin conditions that are not of rheumatic origin, such as psoriasis, inwhich reducing the inflammation resulting from the over production ofprostaglandins could provide a direct benefit. (Fogh et al. (1993) ActaDerm Venerologica 73:191-3). Simply stated COX inhibitors are useful forthe treatment of symptoms of chronic inflammatory diseases, as well as,the occasional ache and pain resulting from transient inflammation.

In addition to their use as anti-inflammatory agents, another potentialrole for COX inhibitors is in the treatment of cancer. Over expressionof COX-2 has been demonstrated in various human malignancies andinhibitors of COX-2 have been shown to be efficacious in the treatmentof animals with skin, breast and bladder tumors. While the mechanism ofaction is not completely defined, the over expression of COX-2 has beenshown to inhibit apoptosis and increase the invasiveness of tumorgeniccell types. (Dempke et al. (2001) J. Can. Res. Clin. Oncol. 127:411-17;Moore and Simmons (2000) Current Med. Chem. 7:1131-44). It is possiblethat enhanced production of prostaglandins resulting from the overexpression of COX-2 promotes cellular proliferation and consequently,increases angiogenesis. (Moore and Simmons (2000) Current Med. Chem.7:1131-44; Fenton et al. (2001) Am. J. Clin. Oncol. 24:453-57).

There have been a number of clinical studies evaluating COX-2 inhibitorsfor potential use in the prevention and treatment of different type ofcancers. Aspirin, a non-specific NSAID, for example, has been found toreduce the incidence of colorectal cancer by 40-50% (Giovannucci et al.(1995) N Engl J Med. 333:609-614) and mortality by 50% (Smalley et al.(1999) Arch Intern Med. 159:161-166). In 1999, the FDA approved theCOX-2 inhibitor CeleCOXib for use in FAP (Familial AdemonatousPolyposis) to reduce colorectal cancer mortality. It is believed thatother cancers, with evidence of COX-2 involvement, may be successfullyprevented and/or treated with COX-2 inhibitors including, but notlimited to esophageal cancer, head and neck cancer, breast cancer,bladder cancer, cervical cancer, prostate cancer, hepatocellularcarcinoma and non-small cell lung cancer. (Jaeckel et al. (2001) Arch.Otolarnygol. 127:1253-59; Kirschenbaum et al. (2001) Urology 58:127-31;Dannhardt and Kiefer (2001) Eur. J. Med. Chem. 36:109-26). COX-2inhibitors may also prove successful in preventing colon cancer inhigh-risk patients. There is also evidence that COX-2 inhibitors canprevent or even reverse several types of life-threatening cancers. Todate, as many as fifty studies show that COX-2 inhibitors can preventpremalignant and malignant tumors in animals, and possibly preventbladder, esophageal and skin cancers as well.

Recent scientific progress has identified correlations between COX-2expression, general inflammation and the pathogenesis of Alzheimer'sDisease (AD). (Ho et al. (2001) Arch. Neurol. 58:487-92). In animalmodels, transgenic mice that over express the COX-2 enzyme have neuronsthat are more susceptible to damage. The National Institute on Aging(NIA) is launching a clinical trial to determine whether NSAIDs can slowthe progression of Alzheimer's Disease. Naproxen (a non-selective NSAID)and rofeCOXib (Vioxx, a COX-2 specific selective NSAID) will beevaluated. Previous evidence has indicated inflammation contributes toAlzheimer's Disease. According to the Alzheimer's Association and theNIA, about 4 million people suffer from AD in the U.S.; and this isexpected to increase to 14 million by mid-century.

The COX enzyme (also known as prostaglandin H2 synthase) catalyzes twoseparate reactions. In the first reaction, arachidonic acid ismetabolized to form the unstable prostaglandin G2 (PGG2), acyclooxygenase reaction. In the second reaction, PGG2 is converted tothe endoperoxide PGH2, a peroxidase reaction. The short-lived PGH2non-enzymatically degrades to PGE2. The compounds described herein arethe result of a discovery strategy that combined an assay focused on theinhibition of COX-1 and COX-2 peroxidase activity with a chemicaldereplication process to identify novel inhibitors of the COX enzymes.

Flavonoids are a widely distributed group of natural products. Theintake of flavonoids has been demonstrated to be inversely related tothe risk of incident dementia. The mechanism of action, while not known,has been speculated as being due to the anti-oxidative effects offlavonoids. (Commenges et al. (2000) Eur. J. Epidemiol 16:357-363).Polyphenol flavones induce programmed cell death, differentiation, andgrowth inhibition in transformed colonocytes by acting at the mRNA levelon genes including COX-2, Nf kappaB and bcl-X(L). (Wenzel et al. (2000)Cancer Res. 60:3823-3831). It has been reported that the number ofhydroxyl groups on the B ring is important in the suppression of COX-2transcriptional activity. (Mutoh et al. (2000) Jnp. J. Cancer Res.91:686-691).

Free-B-Ring flavones and flavonols are a specific class of flavonoids,which have no substituent groups on the aromatic B ring, as illustratedby the following general structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ^(+X) ⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc. Free-B-Ringflavonoids are relatively rare. Out of a total 9396 flavonoidssynthesized or isolated from natural sources, only 231 Free-B-Ringflavonoids are known. (The Combined Chemical Dictionary, Chapman &Hall/CRC, Version 5:1 June 2001).

The Chinese medicinal plant, Scutellaria baicalensis containssignificant amounts of Free-B-Ring flavonoids, including baicalein,baicalin, wogonin and baicalenoside. Traditionally, this plant has beenused to treat a number of conditions including clearing away heat,purging fire, dampness-warm and summer fever syndromes; polydipsiaresulting from high fever; carbuncle, sores and other pyogenic skininfections; upper respiratory infections, such as acute tonsillitis,laryngopharyngitis and scarlet fever; viral hepatitis; nephritis;pelvitis; dysentery; hematemesis and epistaxis. This plant has alsotraditionally been used to prevent miscarriage. (See Encyclopedia ofChinese Traditional Medicine, ShangHai Science and Technology Press,ShangHai, China, 1998). Clinically Scutellaria is now used to treatconditions such as pediatric pneumonia, pediatric bacterial diarrhea,viral hepatitis, acute gallbladder inflammation, hypertension, topicalacute inflammation, resulting from cuts and surgery, bronchial asthmaand upper respiratory infections (Encyclopedian of Chinese TraditionalMedicine, ShangHai Science and Technology Press, ShangHai, China, 1998).The pharmacological efficacy of Scutellaria roots for treating bronchialasthma is reportedly related to the presence of Free-B-Ring flavonoidsand their suppression of eotaxin associated recruitment of eosinophils.(Nakajima et al. (2001) Planta Med. 67(2):132-135).

Free-B-Ring flavonoids have been reported to have diverse biologicalactivity. For example, galangin (3,5,7-trihydroxyflavone) acts asanti-oxidant and free radical scavenger and is believed to be apromising candidate for anti-genotoxicity and cancer chemoprevention.(Heo et al. (2001) Mutat. Res. 488(2):135-150). It is an inhibitor oftyrosinase monophenolase (Kubo et al. (2000) Bioorg. Med. Chem.8(7):1749-1755), an inhibitor of rabbit heart carbonyl reductase(Imamura et al. (2000) J. Biochem. 127(4):653-658), has antimicrobialactivity (Afolayan and Meyer (1997) Ethnopharmacol. 57(3):177-181) andantiviral activity (Meyer et al. (1997) J. Ethnopharmacol.56(2):165-169). Baicalein and galangin, two other Free-B-Ringflavonoids, have antiproliferative activity against human breast cancercells. (So et al. (1997) Cancer Lett. 112(2):127-133).

Typically, flavonoids have been tested for activity randomly based upontheir availability. Occasionally, the requirement of substitution on theB-ring has been emphasized for specific biological activity, such as theB-ring substitution required for high affinity binding to p-glycoprotein(Boumendjel et al. (2001) Bioorg. Med. Chem. Lett. 11(1):75-77);cardiotonic effect (Itoigawa et al. (1999) J. Ethnopharmacol. 65(3):267-272), protective effect on endothelial cells against linoleic acidhydroperoxide-induced toxicity (Kaneko and Baba (1999) BiosciBiotechnol. Biochem 63(2):323-328), COX-1 inhibitory activity (Wang(2000) Phytomedicine 7:15-19) and prostaglandin endoperoxide synthase(Kalkbrenner et al. (1992) Pharmacology 44(1):1-12). Only a fewpublications have mentioned the significance of the unsubstituted B ringof the Free-B-Ring flavonoids. One example, is the use of 2-phenylflavones, which inhibit NAD(P)H quinone acceptor oxidoreductase, aspotential anticoagulants. (Chen et al. (2001) Biochem. Pharmacol.61(11):1417-1427).

The reported mechanism of action related to the anti-inflammatoryactivity of various Free-B-Ring flavonoids has been controversial. Theanti-inflammatory activity of the Free-B-Ring flavonoids, chrysin (Lianget al. (2001) FEBS Lett. 496(1):12-18), wogonin (Chi et al. (2001)Biochem. Pharmacol. 61:1195-1203) and halangin (Raso et al. (2001) LifeSci. 68(8):921-931), has been associated with the suppression ofinducible cyclooxygenase and nitric oxide synthase via activation ofperoxisome-proliferator activated receptor gamma and influence ondegranulation and AA release. (Tordera et al. (1994) Z. Naturforsch [C]49:235-240). It has been reported that oroxylin, baicalein and wogonininhibit 12-lipoxygenase activity without affecting cyclooxygenase. (Youet al. (1999) Arch. Pharm. Res. 22(1):18-24). More recently, theanti-inflammatory activity of wogonin, baicalin and baicalein has beenreported as occurring through inhibition of inducible nitric oxidesynthase and COX-2 gene expression induced by nitric oxide inhibitorsand lipopolysaccharide. (Chen et al. (2001) Biochem. Pharmacol.61(11):1417-1427). It has also been reported that oroxylin acts viasuppression of nuclear factor-kappa B activation. (Chen et al. (2001)Biochem. Pharmacol. 61(11):1417-1427). Finally, wogonin reportedlyinhibits inducible PGE2 production in macrophages. (Wakabayashi andYasui (2000) Eur. J. Pharmacol. 406(3):477-481). Inhibition of thephosphorylation of mitrogen-activated protein kinase and inhibition ofCa²⁺ ionophore A23187 induced prostaglandin E2 release by baicalein hasbeen reported as the mechanism of anti-inflammatory activity ofScutellariae Radix. (Nakahata et al. (1999) Nippon Yakurigaku Zasshi,114, Supp. 11:215P-219P; Nakahata et al. (1998) Am. J. Chin Med.26:311-323). Baicalin from Scutellaria baicalensis, reportedly inhibitssuperantigenic staphylococcal exotoxins stimulated T-cell proliferationand production of IL-1β, interleukin 6 (IL-6), tumor necrosis factor-α(TNF-α), and interferon-γ (IFN-γ). (Krakauer et al. (2001) FEBS Lett.500:52-55). Thus, the anti-inflammatory activity of baicalin has beenassociated with inhibiting the proinflammatory cytokines mediatedsignaling pathways activated by superantigens. However, it has also beenproposed that the anti-inflammatory activity of baicalin is due to thebinding of a variety of chemokines, which limits their biologicalactivity. (Li et al. (2000) Immunopharmacology 49:295-306). Recently,the effects of baicalin on adhesion molecule expression induced bythrombin and thrombin receptor agonist peptide (Kimura et al. (2001)Planta Med. 67:331-334), as well as, the inhibition of mitogen-activatedprotein kinase cascade (MAPK) (Nakahata et al. (1999) Nippon YakurigakuZasshi, 114, Supp 11:215P-219P; Nakahata et al. (1998) Am. J. Chin Med.26:311-323) have been reported. To date there have been no reports thatlink Free-B-Ring flavonoids with COX-2 inhibitory activity.

To date, a number of naturally occurring Free-B-Ring flavonoids havebeen commercialized for varying uses. For example, liposome formulationsof Scutellaria extracts have been utilized for skin care (U.S. Pat. Nos.5,643,598; 5,443,983). Baicalin has been used for preventing cancer, dueto its inhibitory effects on oncogenes (U.S. Pat. No. 6,290,995).Baicalin and other compounds have been used as antiviral, antibacterialand immunomodulating agents (U.S. Pat. No. 6,083,921) and as naturalanti-oxidants (Poland Pub. No. 9,849,256). Chrysin has been used for itsanxiety reducing properties (U.S. Pat. No. 5,756,538). Anti-inflammatoryflavonoids are used for the control and treatment of anorectal andcolonic diseases (U.S. Pat. No. 5,858,371), and inhibition oflipoxygenase (U.S. Pat. No. 6,217,875). Flavonoid esters constituteactive ingredients for cosmetic compositions (U.S. Pat. No. 6,235,294).

Japanese Patent No. 63027435, describes the extraction, and enrichmentof baicalein and Japanese Patent No. 61050921 describes the purificationof baicalin.

SUMMARY OF THE INVENTION

The present invention includes methods that are effective in inhibitingthe cyclooxygenase enzyme COX-2. The method for inhibiting thecyclooxygenase enzyme COX-2 is comprised of administering a compositioncomprising a Free-B-Ring flavonoid or a composition containing a mixtureof Free-B-Ring flavonoids to a host in need thereof.

The present also includes methods for the prevention and treatment ofCOX-2 mediated diseases and conditions. The method for preventing andtreating COX-2 mediated diseases and conditions is comprised ofadministering to a host in need thereof an effective amount of acomposition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids and a pharmaceuticallyacceptable carrier.

The Free-B-Ring flavonoids that can be used in accordance with thefollowing include compounds illustrated by the following generalstructure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

The method of this invention can be used to treat and prevent a numberof COX-2 mediated diseases and conditions including, but not limited to,osteoarthritis, rheumatoid arthritis, menstrual cramps, systemic lupuserythromatosus, psoriasis, chronic tension headaches, migraineheadaches, topical wound and minor inflammatory conditions, inflammatorybowel disease and solid cancers.

The Free-B-Ring flavonoids of this invention may be obtained bysynthetic methods or extracted from the family of plants including, butnot limited to Annonaceae, Asteraceae, Bignoniaceae, Combretaceae,Compositae, Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae,Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae and Zingiberacea. TheFree-B-Ring flavonoids can be extracted, concentrated, and purified fromthe following genus of high plants, including but not limited to Desmos,Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium,Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria,Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus,Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.

The compositions of this invention can be administered by any methodknown to one of ordinary skill in the art. The modes of administrationinclude, but are not limited to, enteral (oral) administration,parenteral (intravenous, subcutaneous, and intramuscular) administrationand topical application. The method of treatment according to thisinvention comprises administering internally or topically to a patientin need thereof a therapeutically effective amount of the individualand/or a mixture of multiple Free-B-Ring flavonoids from a single sourceor multiple sources that include, but not limited to, syntheticallyobtained, naturally occurring, or any combination thereof.

This invention includes an improved method for isolating and purifyingFree-B-Ring flavonoids from plants containing these compounds. Themethod of the present invention comprises: a) extracting the groundbiomass of a plant containing Free-B-Ring flavonoids; b) neutralizingand concentrating said extract; and c) purifying said neutralized andconcentrated extract. In a preferred embodiment of the invention theextract is purified using a method selected from the group consisting ofrecrystallization, precipitation, solvent partition and/orchromatographic separation. The present invention provides acommercially viable process for the isolation and purification ofFree-B-Ring flavonoids having desirable physiological activity.

The present invention implements a strategy that combines a series ofbiomolecular screens with a chemical dereplication process to identifyactive plant extracts and the particular compounds within those extractsthat specifically inhibit COX-2 enzymatic activity and inflammation. Atotal of 1230 plant extracts were screened for their ability to inhibitthe peroxidase activity associated with recombinant COX-2. This primaryscreen identified 22 plant extracts that were further studied for theirability to specifically and selectively inhibit COX-2 in vitro in bothcell based and whole blood assays. Those extracts that were efficaciousin vitro were then tested for their ability to inhibit inflammation invivo using a both air pouch and topical ear-swelling models ofinflammation when administered by multiple routes (IP and oral). Thesestudies resulted in the discovery of botanical extracts that inhibitedCOX-2 activity and were efficacious both in vitro and in vivo. Thesestudies also resulted in the identification of specific Free-B-Ringflavonoids associated with COX-2 inhibition in each of these extracts.Applicant believes that this is first report of a correlation betweenFree-B-Ring flavonoid structure and COX-2 inhibitory activity.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts graphically the inhibition of COX-1 and COX-2 by HTPfractions from Scutellaria baicaensis. The extracts were prepared andfractionated as described in Examples 1 and 3. The extracts wereexamined for their inhibition of the peroxidase activity of recombinantovine COX-1 (▪) or ovine COX-2 (♦). The data is presented as percent ofuntreated control.

FIG. 2 depicts the high pressure liquid chromatography (HPLC)chromatograms of Free-B-Ring Flavonoids in organic extracts fromScutellaria lateriflora roots (FIG. 2A), Scutellaria orthocalyx roots(FIG. 2B) and Scutellaria baicaensis roots (FIG. 2C).

FIG. 3 demonstrates the in vivo efficacy of Free-B-Ring Flavonoids fromScutellaria baicaensis on arachidonic acid induced inflammation. The invivo efficacy was evaluated based on the ability to inhibit swellinginduced by direct application of arachidonic acid as described inExample 9. The average differences in swelling between the treated earsand control ears are represented in FIG. 3A. FIG. 3B demonstrates thepercent inhibition of each group in comparison to the arachidonic acidtreated control.

FIG. 4 illustrates the in vivo efficacy of Free-B-Ring Flavonoidsisolated from Scutellaria baicalensis on inflammation induced byZymosan. Zymosan was used to elicit a pro-inflammatory response in anair pouch as described in Example 9. Markers of inflammation includinginfiltration of pro-inflammatory cells (FIG. 4A), percent inhibition ofMPO activity with in the air pouch fluid (FIG. 4B), and percentinhibition of TNF-α production (FIG. 4C) were used to evaluate theefficacy and mechanism of action of the anti-inflammatory activity ofthe Free-B-Ring Flavonoids from Scutellaria baicalensis.

FIG. 5 illustrates graphically the % change in composite WOMAC indexscores following 60 days of treatment with placebo, celebrex at a dosageof 200 mg/day, Univestin at a dosage of 250 mg/day and Univestin at adosage of 500 mg/day as described in Example 11.

FIG. 6 illustrates graphically the % change in WOMAC index scores ofstiffness following 60 days of treatment with placebo, celebrex at adosage of 200 mg/day, Univestin at a dosage of 250 mg/day and Univestinat a dosage of 500 mg/day as described in Example 11.

FIG. 7 illustrates graphically the % change in WOMAC index scoresrelated to physical function following 60 days of treatment withplacebo, celebrex at a dosage of 200 mg/day, Univestin at a dosage of250 mg/day and Univestin at a dosage of 500 mg/day as described inExample 11.

FIG. 8 illustrates graphically the % change in WOMAC index scoresrelated to pain following 60 days of treatment with placebo, celebrex ata dosage of 200 mg/day, Univestin at a dosage of 250 mg/day andUnivestin at a dosage of 500 mg/day as described in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

“Free-B-Ring Flavonoids” as used herein are a specific class offlavonoids, which have no substituent groups on the aromatic B ring, asillustrated by the following general structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans, as well as, other animals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the alleviation of the signs, symptoms or causes of adisease or any other desirous alteration of a biological system.

A “host” is a living subject, human or animal, into which thecompositions described herein are administered.

Note, that throughout this application various citations are provided.Each citation is specifically incorporated herein in its entirety byreference.

The present invention includes methods that are effective in inhibitingthe cyclooxygenase enzyme COX-2. The method for inhibiting thecyclooxygenase enzyme COX-2 is comprised of administering a compositioncomprising a Free-B-Ring flavonoid or a composition containing a mixtureof Free-B-Ring flavonoids to a host in need thereof.

The present also includes methods for the prevention and treatment ofCOX-2 mediated diseases and conditions. The method for preventing andtreating COX-2 mediated diseases and conditions is comprised ofadministering to a host in need thereof an effective amount of acomposition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids and a pharmaceuticallyacceptable carrier.

The Free-B-Ring flavonoids that can be used in accordance with thefollowing include compounds illustrated by the general structure setforth above. The Free-B-Ring flavonoids of this invention may beobtained by synthetic methods or may be isolated from the family ofplants including, but not limited to Annonaceae, Asteraceae,Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae,Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae,Sinopteridaceae, Ulmaceae, and Zingiberacea. The Free-B-Ring flavonoidscan be extracted, concentrated, and purified from the following genus ofhigh plants, including but not limited to Desmos, Achyrocline, Oroxylum,Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea,Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys,Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza,Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma,Notholaena, Pinus, Ulmus, and Alpinia.

The flavonoids can be found in different parts of plants, including butnot limited to stems, stem barks, twigs, tubers, roots, root barks,young shoots, seeds, rhizomes, flowers and other reproductive organs,leaves and other aerial parts.

In order to identify compounds able to inhibit the COX enzymes anextract library composed of 1230 extracts from 615 medicinal plantscollected from China, India, and other countries was created. A generalmethod for preparing the extracts is described in Example 1, which usesthe Scutellaria species for purposes of illustration. The extractionprocess yields an organic and an aqueous extract for each speciesexamined. The results of the extraction of various Scutellaria speciesare set forth in Table 1. These primary extracts are the source materialused in the preliminary assay to identify inhibitors of thecyclooxygenase enzyme's peroxidase activity, which is one of the mainfunctional activities of cyclooxygenase and is responsible forconverting PGG2 to PGH2 and ultimately PGE2, as described in detailabove.

This assay is described in Example 2 and the results are set forth inTable 2. With reference to Table 2, it can be seen that two species ofScutellaria and three other plant species, all of which containFree-B-ring flavonoids as common components, showed inhibitory activityin the primary screen against the peroxidase activity of COX-2 albeit todiffering degrees. The COX-2 inhibitory activity is found predominantlyin the organic extracts, which contain the most of medium polarityFree-B-Ring flavonoids.

The COX-2 inhibitory activity from the primary assay of the crudeextracts has been confirmed by measurement of dose response and IC₅₀(the concentration required to inhibit 50% of the enzyme's activity).The IC₅₀ values are set forth in Table 3. As can be seen in Table 3, inthis assay Scutellaria orthocalyx root extract and Murica nana leafextract were the most efficacious (IC₅₀=6-10 mg/mL). Extracts fromScutellaria sp. that demonstrated the greatest selectivity against COX-2were Scutellaria lateriflora (COX-2 IC₅₀: 30 mg/mL; COX-1 IC₅₀: 80mg/mL). Thus, the primary screen for inhibitors of the COX enzymeidentified five extracts containing Free-B-Ring flavonoids that wereefficacious in vitro and some of which demonstrated specificity for theCOX-2 enzyme.

In order to efficiently identify active compounds from plant extracts, ahigh throughput fractionation process was used, as described in Example3. Briefly, the active organic and aqueous extracts were fractionatedusing two different methodologies, respectively. The fractions werecollected in a 96 deep well plate. Each of the fractions was tested forits ability to inhibit COX activity as per the primary assay, asdescribed in Example 4. The results are set forth in FIG. 1, whichdepicts the profile of COX-1 and COX-2 inhibition by various HTPfractions derived from the roots of Scutellaria baicalensis. As can beseen in FIG. 1, the most potent COX inhibitory activity was found in twomajor fractions, E11 and F11. It should be noted that three separate HTPfractions actually exhibit inhibitory activity, suggesting that thereare multiple compounds contributing to the observed inhibitory effectsof the whole extract.

The separation, purification and identification of the activeFree-B-Ring flavonoids present in the organic extract of Scutellariaorthocalyx is described in Example 5. Using the methodology described inExample 5, baicalein was identified as the major active component in theorganic extract from the roots of Scutellaria orthocalyx. As shown inthe Example 6, several other Free-B-Ring flavonoids have been isolatedand tested for inhibition of COX-1 and COX-2 enzymatic activity. Theresults are set forth in Table 4.

Example 7 and Table 5 set forth the content and quantity of theFree-B-Ring flavonoids in five active plant extracts from threedifferent species of plants. The Free-B-Ring flavonoids are present inmuch greater amounts in the organic extracts verses the aqueousextracts. This explains why the COX-2 inhibitory activity has usuallyshown up in the organic extracts rather than the aqueous extracts.

The primary assay described in Example 2 to identify active extracts isa cell free system utilizing recombinant enzymes. To further demonstratethe biological activity of the active extracts and compounds, two modelsthat represent cell based in vitro efficacy and animal based in vivoefficacy were employed. The method used to evaluate in vitro efficacyand selectivity is described in Example 8. Two cell lines were selectedthat could be induced to express primarily COX-1 (THP-1 cells) and COX-2(HOSC cells), respectively. Each cell type was examined for theproduction of PGE2, the primary product of the COX enzymes. The resultsare set forth in Table 6, which shows that three organic extracts fromthree different species of Scutellaria showed inhibition of both theCOX-1 and COX-2 enzymes with a preference for the COX-1 enzyme. Whilethe use of the THP-1 cell line is important and demonstrates the abilityof the active compounds to cross the cell membrane, it is animmortalized cell line, therefore evaluation of the efficacy ofFree-B-Ring flavonoids based on a more relevant model system isdesirable. As a result, the extracts were also evaluated using wholeblood as the primary source of both COX-1 and COX-2 activity. Thissystem measures the inhibition of the production of PGE2 vs. TXB₂ todifferentiate between COX-2 and COX-1 inhibitory activity, respectively.The results, which are set forth in Table 6 demonstrate that both theCOX-1 and COX-2 enzymes are inhibited by the Free-B-Ring flavonoids fromall three Scutellaria root extracts. The IC₅₀ values suggest that withinthis system all Free-B-Ring flavonoids, except those from Scutellariabaicaensis are more efficacious against COX-2. Taken as a whole, theinhibitory effect of the active compounds within these extracts issignificant and efficacious in both cell free and cell-based systems invitro. Also, no cell toxicity been observed in the testing process.

Two separate in vivo models were employed to determine whether the invitro efficacy observed from the Free-B-Ring flavonoids translated to anability to inhibit in vivo inflammatory responses. The two models aredescribed in Example 9. The first of these systems was designed tomeasure inflammation resulting directly from the arachidonic acidmetabolism pathway. In this example, mice were treated with Free-B-Ringflavonoids from three Scutellaria species prior to the topicalapplication of AA to the ear to induce the inflammatory response. Theeffect of pretreating the animals was then measured by the inhibition ofthe ear swelling using a micrometer. The Free-B-Ring flavonoidscontaining extracts from these three Scutellaria species demonstratedvarying degrees of efficacy. For example, the Free-B-Ring flavonoidsextracted from the roots of Scutellaria baicaensis inhibited earswelling by 60% in comparison to controls when delivered by both oraland IP routes as illustrated in FIGS. 3A and B. This is the similar tothe degree of inhibition seen with the positive control indomethacin,when delivered IP at a concentration of 5 mg/kg. Free-B-Ring flavonoidsextracted from Scutellaria orthocalyx were efficacious when delivered byIP routes of administration, but had no effect when delivered by oralroutes and finally the Free-B-Ring flavonoids extracted from Scutellarialateriflora showed no effect regardless of the route of administration(data not shown).

The Free-B-Ring flavonoids isolated from Scutellaria baicaensis havebeen clearly demonstrated to be the most efficacious againstinflammation induced directly by the arachidonic acid. Therefore, theefficacy of these Free-B-Ring flavonoids was examined using a secondmodel in which multiple mechanisms of inflammation are ultimatelyresponsible for the final effect. This system is therefore more relevantto naturally occurring inflammatory responses. Using this model, a verypotent activator of the complement system is injected into an air pouchcreated on the back of Balb/C mice. This results in a cascade ofinflammatory events including, infiltration of inflammatory cells,activation of COX enzymes, resulting in the release of PGE₂, the enzymemyeloperoxidase (MPO), and production of a very specific profile ofpro-inflammatory cytokines including TNF-α. These studies demonstratedthat even though the Free-B-Ring flavonoids isolated from Scutellariabaicaensis did not inhibit the initial infiltration (chemotacticresponse) of inflammatory cells into the air pouch, they blocked theactivation of those cells. This is evidenced by the lack of MPO excretedinto the extracellular fluid of the pouch and the noted lack ofproduction of TNF-α. The results are set forth in FIG. 4. The datademonstrates that the Free-B-Ring flavonoids are efficacious and helpcontrol an inflammatory response in a model system where multipleinflammatory pathways are active.

The preparation of products for administration in pharmaceuticalpreparations may be performed by a variety of methods well known tothose skilled in the art. The Free-B-Ring flavonoids may be formulatedas an herb powder in the form of its natural existence; as solventand/or supercritical fluid extracts in different concentrations; asenriched and purified compounds through recrystallization, columnseparation, solvent partition, precipitation and other means, as a pureand/or a mixture containing substantially purified Free-B-Ringflavonoids prepared by synthetic methods.

Various delivery systems are known in the art and can be used toadminister the therapeutic compositions of the invention, e.g., aqueoussolution, encapsulation in liposomes, microparticles, and microcapsules.

Therapeutic compositions of the invention may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and Free-B-Ring flavonoid(s) constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder; or directly capsulated and/or tabletedwith other inert carriers for oral administration. Such formulations maybe stored either in a ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing the compositions for systemic delivery may bevia enteral, subcutaneous, intramuscular, intravenous, intranasal orvaginal or rectal suppository.

The amount of the composition that will be effective in the treatment ofa particular disorder or condition will depend on the nature of thedisorder of condition, which can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness or advancement of the disease orcondition, and should be decided according to the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curved derived from in vitro or animal model test systems.For example, an effective amount of the composition of the invention isreadily determined by administering graded doses of the composition ofthe invention and observing the desired effect.

The method of treatment according to this invention comprisesadministering internally or topically to a patient in need thereof atherapeutically effective amount of the individual and/or a mixture ofmultiple Free-B-Ring flavonoids from a single source or multiplesources. The purity of the individual and/or a mixture of multipleFree-B-Ring flavonoids includes, but is not limited to 0.01% to 100%,depending on the methodology used to obtain the compound(s). In apreferred embodiment doses of the Free-B-Ring flavonoids andpharmaceutical compositions containing that same are an efficacious,nontoxic quantity generally selected from the range of 0.01 to 200 mg/kgof body weight. Persons skilled in the art using routine clinicaltesting are able to determine optimum doses for the particular ailmentbeing treated.

This invention includes an improved method for isolating and purifyingFree-B-Ring flavonoids from plants, which is described in Example 10. InExample 10, Free-B-Ring flavonoids from two Scutellaria species wereextracted with different solvent systems. The results are set forth inTables 7 and 8. The improved method of this invention comprises:extraction of the ground biomass of a plant containing Free-B-Ringflavonoids with single or combination of organic solvent and/or water;neutralization and concentration of the neutralized extract; andpurification of said extract by recrystallization and/or chromatography.As provided above, these Free-B-Ring flavonoids can be isolated from thegenera of more than twenty plant families. The method of this inventioncan be extended to the isolation of these compounds from any plantsource containing these compounds.

Additionally the Free-B-Ring flavonoids can be isolated from variousparts of the plant including, but not limited to, the whole plant,stems, stem bark, twigs, tubers, flowers, fruit, roots, root barks,young shoots, seeds, rhizomes and aerial parts. In a preferredembodiment the Free-B-Ring flavonoids are isolated from the roots,reproductive organs or the whole plant.

The solvent used for extraction of the ground biomass of the plantincludes, but is not limited to water, acidified water, water incombination with miscible hydroxylated organic solvent(s) including, butnot limited to, methanol or ethanol and an mixture of alcohols withother organic solvent(s) such as THF, acetone, ethyl acetate etc. In oneembodiment the extract is neutralized to a pH of 4.5-5.5 and thenconcentrated and dried to yield a powder. The Free-B-Ring flavonoids canthen be purified by various methods including, but not limited torecrystallization, solvent partition, precipitation, sublimation, and/orchromatographic methods including, but not limited to, ion exchangechromatography, absorption chromatography, reverse phase chromatography,size exclusive chromatography, ultra-filtration or a combination ofthereof.

Example 11 describes a clinical study performed to evaluate the efficacyof free-B-ring flavonoids on the relief of pain caused by rheumatoidarthritis or osteoarthritis of the knee and/or hip. The study was asingle-center, randomized, double-blind, placebo-controlled study. Sixtysubjects (n=60) with rheumatoid arthritis or osteoarthritis of the kneeand/or hip were randomly placed into four groups and treated for 60 dayswith a placebo, Univestin (250 mg/day or 500 mg/day) or Celebrex (200mg/day). The Univestin consisted of a proprietary ratio of standardizedextract of Scutellaria baicalensis Georgi with a Baicalin content of62.5% (w/w) and total Free-B-Ring Flavonoids >75% (w/w). Celebrex is atrade name for a prescription drug that is a COX-2 selective inhibitor.Table 9 sets forth the WOMAC index scores before treatment (baselinescores) and Table 10 sets forth the changes in WOMAC index scores aftertreatment. FIGS. 5 to 8 illustrate the results of the study graphically.

As shown in the FIGS. 5 to 8, the WOMAC composite scores and individualsubscores, related to pain, stiffness and physical function exhibitedsignificant improvements after administration of free-B-ring flavonoidscompared to the placebo group. Further, free-B-ring flavonoids exhibitedthe same effectiveness on pain relieve and improvement of physicalfunction as the prescription drug Celebrex. Finally no difference ineffectiveness was observed for the free-B-ring flavonoids at the twodifferent dosages administered.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Organic and Aqueous Extracts fromScutellaria Plants

Plant material from Scutellaria orthocalyx roots, Scutellaria baicaensisroots or Scutellaria lateriflora whole plant was ground to a particlesize of no larger than 2 mm. Dried ground plant material (60 g) was thentransferred to an Erlenmeyer flask and methanol:dichloromethane (1:1)(600 mL) was added. The mixture was shaken for one hour, filtered andthe biomass was extracted again with methanol:dichloromethane (1:1) (600mL). The organic extracts were combined and evaporated under vacuum toprovide the organic extract (see Table 1 below). After organicextraction, the biomass was air dried and extracted once with ultra purewater (600 mL). The aqueous solution was filtered and freeze-dried toprovide the aqueous extract (see Table 1 below).

TABLE 1 Yield of Organic and Aqueous Extracts of various Scutellariaspecies Plant Source Amount Organic Extract Aqueous Extract Scutellariaorthocalyx roots 60 g 4.04 g 8.95 g Scutellaria baicaensis roots 60 g9.18 g 7.18 g Scutellaria lateriflora 60 g 6.54 g 4.08 g (whole plant)

Example 2 Inhibition of COX-2 and COX-1 Peroxidase Activity by PlantExtracts from Various Scutellaria Species

The bioassay directed screening process for the identification ofspecific COX-2 inhibitors was designed to assay the peroxidase activityof the enzyme as described below.

Peroxidase Assay. The assay to detect inhibitors of COX-2 was modifiedfor a high throughput platform (Raz). Briefly, recombinant ovine COX-2(Cayman) in peroxidase buffer (100 mM, TBS, 5 mM EDTA, 1 μM Heme, 0.01mg epinephrine, 0.094% phenol) was incubated with extract (1:500dilution) for 15 minutes. Quantablu (Pierce) substrate was added andallowed to develop for 45 minutes at 25° C. Luminescence was then readusing a Wallac Victor 2 plate reader. The results are set forth in Table2.

Table 2 sets forth the inhibition of enzyme by the organic and aqueousextracts obtained from five plant species, including the roots of twoScutellaria species and extracts from three other plant species, whichare comprised of structurally similar Free-B-Ring Flavonoids. Data ispresented as the percent of peroxidase activity relative to therecombinant ovine COX-2 enzyme and substrate alone. The percentinhibition by the organic extract ranged from 30% to 90%.

TABLE 2 Inhibition of COX-2 Peroxidase activity by Scutellaria speciesInhibition of COX-2 Inhibition of COX-2 Plant Source by organic extractby aqueous extract Scutellaria orthocalyx (root) 55% 77% Scutellariabaicaensis (root) 75% 0% Desmodium sambuense 55% 39% (whole plant)Eucaluptus globulus (leaf) 30% 10% Murica nana (leaf) 90% 0%

Comparison of the relative inhibition of the COX-1 and COX-2 isoformsrequires the generation of IC₅₀ values for each of these enzymes. TheIC₅₀ is defined as the concentration at which 50% inhibition of enzymeactivity in relation to the control is achieved by a particularinhibitor. In the instant case, IC₅₀ values were found to range from 6to 50 μg/mL and 7 to 80 μg/mL for the COX-2 and COX-1 enzymes,respectively, as set forth in Table 3. Comparison, of the IC₅₀ values ofCOX-2 and COX-1 demonstrates the specificity of the organic extractsfrom various plants for each of these enzymes. The organic extract ofScutellaria lateriflora for example, shows preferential inhibition ofCOX-2 over COX-1 with IC₅₀ values of 30 and 80 μg/mL, respectively.While some extracts demonstrate preferential inhibition of COX-2, othersdo not. Examination of the HTP fractions and purified compounds fromthese fractions is necessary to determine the true specificity ofinhibition for these extracts and compounds.

TABLE 3 IC₅₀ Values for Human and Ovine COX-2 and COX-1 IC₅₀ Human IC₅₀Ovine IC₅₀ Ovine COX-2 COX-2 COX-1 Plant Source (μg/mL) (μg/mL) (μg/mL)Scutellaria orthocalyx ND 10 10 (root) Scutellaria baicaensis 30 20 20(root) Scutellaria lateriflora 20 30 80 (whole plant) Eucaluptusglobulus ND 50 50 (leaf) Murica nana  5 6 7 (leaf)

Example 3 HTP Fractionation of Active Extracts

Organic extract (400 mg) from Scutellaria baicaensis roots was loadedonto a prepacked flash column. (2 cm ID×8.2 cm, 10 g silica gel). Thecolumn was eluted using a Hitachi high throughput purification (HTP)system with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B)methanol from 100% A to 100% B in 30 minutes at a flow rate of 5 mL/min.The separation was monitored using a broadband wavelength UV detectorand the fractions were collected in a 96-deep-well plate at 1.9 mL/wellusing a Gilson fraction collector. The sample plate was dried under lowvacuum and centrifugation. DMSO (1.5 mL) was used to dissolve thesamples in each cell and a portion (100 μL was taken for the COXinhibition assay.

Aqueous extract (750 mg) from Scutellaria baicaensis roots was dissolvedin water (5 mL), filtered through a 1 μm syringe filter and transferredto a 4 mL HPLC vial. The solution was then injected by an autosampleronto a prepacked reverse phase column (C-18, 15 μm particle size, 2.5 cmID×10 cm with precolumn insert). The column was eluted using a Hitachihigh throughput purification (HTP) system with a gradient mobile phaseof (A) water and (B) methanol from 100% A to 100% B in 20 minutes,followed by 100% methanol for 5 minutes at a flow rate of 10 mL/min. Theseparation was monitored using a broadband wavelength UV detector andthe fractions were collected in a 96-deep-well plate at 1.9 mL/wellusing a Gilson fraction collector. The sample plate was freeze-dried.Ultra pure water (1.5 mL) was used to dissolve samples in each cell anda portion of 100 μL was taken for the COX inhibition assay.

Example 4 Inhibition of COX Peroxidase Activity by HTP Fractions fromVarious Scutellaria Species

Individual bioactive organic extracts were further characterized byexamining each of the HTP fractions for the ability to inhibit theperoxidase activity of both COX-1 and COX-2 recombinant enzymes. Theresults are depicted in FIG. 1, which depicts the inhibition of COX-2and COX-1 activity by HTP fractions from Scutellaria baicaensis isolatedas described in Example 1 and 3. The profile depicted in FIG. 1 shows apeak of inhibition that is very selective for COX-2. Other Scutellariasp. including Scutellaria orthocalyx and Scutellaria lateriflorademonstrate a similar peak of inhibition (data not shown). However, boththe COX-1 and COX-2 enzymes demonstrate multiple peaks of inhibitionsuggesting that there is more than one molecule contributing to theinitial inhibition profiles.

Example 5 Isolation and Purification of the Active Free-B-RingFlavonoids from the Organic Extract of Scutellaria orthocalyx

The organic extract (5 g) from the roots of Scutellaria orthocalyx,isolated as described in Example 1, was loaded onto prepacked flashcolumn (120 g silica, 40 μm particle size 32-60 μm, 25 cm×4 cm) andeluted with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B)methanol from 100% A to 100% B in 60 minutes at a flow rate of 15mL/min. The fractions were collected in test tubes at 10 mL/fraction.The solvent was evaporated under vacuum and the sample in each fractionwas dissolved in 1 mL of DMSO and an aliquot of 20 μL was transferred toa 96 well shallow dish plate and tested for COX inhibitory activity.Based on the COX assay results, active fractions #31 to #39 werecombined and evaporated. Analysis by HPLC/PDA and LC/MS showed a majorcompound with a retention times of 8.9 minutes and a MS peak at 272 m/e.The product was further purified on a C18 semi-preparation column (25cm×1 cm), with a gradient mobile phase of (A) water and (B) methanol,over a period of 45 minutes at a flow rate of 5 mL/minute. Eighty eightfractions were collected to yield 5.6 mg of light yellow solid. Puritywas determined by HPLC/PDA and LC/MS, and comparison with standards andNMR data. ¹H NMR: δppm. (DMSO-d6) 8.088 (2H, m, H-3′,5′), 7.577 (3H, m,H-2′,4′,6′), 6.932 (1H, s, H-8), 6.613 (1H, s, H-3). MS: [M+1]+=271 m/e.The compound has been identified as Baicalein. The IC₅₀ of Baicaleinagainst the COX-2 enzyme is 10 μg/mL.

Example 6 COX Inhibition of Purified Free-B-Ring Flavonoids

Several other Free-B-Ring Flavonoids have been obtained and tested at aconcentration of 20 μg/mL for COX-2 inhibition activities using themethods described above. The results are summarized in Table 4.

TABLE 4 Inhibition of COX Enzymatic Activity by Purified Free-B-RingFlavonoids Free-B-Ring Flavonoids Inhibition of COX-1 Inhibition ofCOX-2 Baicalein 107% 109% 5,6-Dihydroxy-7- 75% 59% methoxyflavone7,8-Dihydroxyflavone 74% 63% Baicalin 95% 97% Wogonin 16% 12%

Example 7 HPLC Quantification of Free-B-Ring Flavonoids in ActiveExtracts from Scutellaria Orthocalyx Roots, Scutellaria baicaensis Rootsand Oroxylum indicum Seeds

The presence and quantity of Free-B-Ring Flavonoids in five activeextracts from three different plant species have been confirmed and areset forth in the Table 5. The Free-B-Ring Flavonoids were quantitativelyanalyzed by HPLC using a Luna C-18 column (250×4.5 mm, 5 μm) using 0.1%phosphoric acid and acetonitrile gradient from 80% to 20% in 22 minutes.The Free-B-Ring Flavonoids were detected using a UV detector at 254 nmand identified based on retention time by comparison with Free-B-RingFlavonoid standards. The HPLC chromatograms are depicted in FIG. 2.

TABLE 5 Free-B-Ring Flavonoid Content in Active Plant Extracts Weight of% Extractible Total amount % Flavonoids in Active Extracts Extract fromBioMass of Flavonoids Extract Scutellaria orthocalyx 8.95 g 14.9%  0.2mg 0.6% (AE)* Scutellaria orthocalyx 3.43 g 5.7% 1.95 mg 6.4% (OE)*Scutellaria baicaensis 7.18 g 12.0% 0.03 mg 0.07%  (AE)* Scutellariabaicaensis 9.18 g 15.3% 20.3 mg 35.5%  (OE)* Oroxylum indicum 6.58 g11.0%  0.4 mg 2.2% (OE)* *AE: Aqueous Extract *OE: Organic Extract

Example 8 In vitro Study of COX Inhibitory Activity of Free-B-RingFlavonoids from Various Scutellaria Species

In vitro efficacy and COX-2 specificity of Free-B-Ring Flavonoidsisolated from various Scutellaria species were tested in cell basedsystems for their ability to inhibit the generation of arachidonic acidmetabolites. Cell lines HOSC that constitutively express COX-2 and THP-1that express COX-1 were tested for their ability to generateprostaglandin E2 (PGE2) in the presence of arachidonic acid.

COX-2 Cell Based Assay. HOSC (ATCC#8304-CRL) cells were cultured to80-90% confluence. The cells were trysinized, washed and resuspended in10 mL at 1×10⁶ cells/mL in tissue culture media (MEM). The cellsuspension (200 μL) was plated out in 96 well tissue culture plates andincubated for 2 hours at 37° C. and 5% CO₂. The media was then replacedwith new HOSC media containing 1 ng/mL IL-1 b and incubated overnight.The media was removed again and replaced with 190 mL HOSC media. Testcompounds were then added in 10 μL of HOSC media and were incubated for15 minutes at 37° C. Arachidonic acid in HOSC media (20 mL, 100 μM) wasadded and the mixture was incubated for 10 minutes on a shaker at roomtemperature. Supernatant (20 μL) was transferred to new platescontaining 190 μL/well of 100 μM indomethacin in ELISA buffer. Thesupernatants were analyzed as described below by ELISA.

COX-1 Cell Based Assay. THP-1 cells were suspended to a volume of 30 mL(5×10⁵ cells/mL). TPA was added to a final concentration of 10 nM TPAand cultured for 48 hours to differentiate cells to macrophage(adherent). The cells were resuspended in HBSS (25 mL) and added to 96well plates in 200 mL volume at 5×10⁵ cells/well. The test compounds inRPMI 1640 (10 μL) were then added and incubated for 15 minutes at 37° C.Arachidonic acid in RPMI (20 μL) was then added and the mixture wasincubated for 10 minutes on a shaker at room temperature. Supernatant(20 μL) was added to Elisa buffer (190 μL) containing indomethacin (100μM). The supernatants were then analyzed by ELISA, as described below.

COX-2 Whole Blood assay. Peripheral blood from normal, healthy donorswas collected by venipuncture. Whole blood (500 μL) was incubated withtest compounds and extracts for 15 minutes at 37° C. Lipopolysaccharide(from E. coli serotype 0111:B4) was added to a final concentration of100 μg/mL and cultured overnight at 37° C. Blood was centrifuged(12,000×g) and the plasma was collected. Plasma (100 μL) was added tomethanol (400 μL) to precipitate proteins. Supernatants were measuredfor PGE2 production by ELISA. This procedure is a modification of themethods described by Brideau et al. (1996) Inflamm. Res. 45:68-74.

COX-1 Whole Blood Assay. Fresh blood was collected in tubes notcontaining anti-coagulants and immediately aliquoted into 500 μLaliquots in siliconized microcentrifuge tubes. Test samples were added,vortexed and allowed to clot for 1 hour at 37° C. Samples were thencentrifuged (12,000×g) and the plasma was collected. The plasma (100 μL)was added to methanol (400 μL) to precipitate proteins. Supernatantswere measured for TXB2 production by ELISA. This procedure is amodification of the methods described by Brideau et al. (1996) Inflamm.Res. 45:68-74.

ELISA Assays. inmunolon-4 ELISA plates were coated with capture antibody0.5-4 μg/mL in carbonate buffer (pH 9.2) overnight at 4° C. The plateswere washed and incubated for 2 hours with blocking buffer (PBS+1% BSA)at room temperature. The plates were washed again and test sample (100μL) was added and incubated for 1 hour at room temperature whileshaking. Peroxidase conjugated secondary antibody was added in a 50 μLvolume containing 0.5-4 mg/mL and incubated for 1 hour at roomtemperature while shaking. The plates were then washed three times andTMB substrate (100 μL) was added. The plates were allowed to develop for30 minutes, after which the reaction was stopped by the addition of 1 Mphosphoric acid (100 μL). The plates were then read at 450 nm using aWallac Victor 2 plate reader.

Cytotoxicity. Cellular cytotoxicity was assessed using a colorimetrickit (Oxford biochemical research) that measures the release of lactatedehydrogenase in damaged cells. Assays were completed according tomanufacturers' directions. No cytotoxicity has been observed for any ofthe tested compounds.

The results of the assays are set forth in Table 6. The data ispresented as IC₅₀ values for direct comparison. With reference to Table6, IC₅₀ values are generally lower for COX-1 than COX-2. Whole blood wasalso measured for the differential inhibition of PGE2 generation (ameasure of COX-2 in this system) or thromboxane B2 (TXB2) (a measure ofCOX-1 activation). Referring to Table 6, these studies clearlydemonstrate specificity for COX-2 inhibition from the organic extractsin two of the three species tested. Possible reasons for thisdiscrepancy are the fundamental differences between immortalized celllines that constitutively express each of the enzymes and primary cellsderived from whole blood that that are induced to express COX enzymes.Primary cells are the more relevant model to study the in vivoinflammation process.

TABLE 6 Inhibition of COX Activity in Whole Cell Systems Cell Line BasedAssay Whole Blood Assay Plant Source IC₅₀ COX-2 IC₅₀ COX-1 IC₅₀ COX-2IC₅₀ COX-1 Scutellaria 50 μg/mL 18 μg/mL 10 μg/mL >50 μg/mL   orthocalyx(root) Scutellaria 82 μg/mL 40 μg/mL 20 μg/mL  8 μg/mL baicaensis (root)Scutellaria 60 μg/mL 30 μg/mL  8 μg/mL 20 μg/mL lateriflora (wholeplant)

Example 9 In vivo Study of COX Inhibitory Activity of Free-B-RingFlavonoids from Various Scutellaria Species

In vivo inhibition of inflammation was measured using two model systems.The first system (ear swelling model) measures inflammation induceddirectly by arachidonic acid. This is an excellent measure of COX-2inhibition, but does not measure any of the cellular events which wouldoccur upstream of arachidonic acid liberation from cell membranephospholipids by phospholipase A2 (PLA2). Therefore, to determine howinhibitors function in a more biologically relevant response the airpouch model was employed. This model utilizes a strong activator ofcomplement to induce an inflammatory response that is characterized by astrong cellular infiltrate and inflammatory mediator productionincluding cytokines as well as arachidonic acid metabolites.

Ear Swelling Model. The ear swelling model is a direct measure of theinhibition of arachidonic acid metabolism. Arachidonic acid in acetoneis applied topically to the ears of mice. The metabolism of arachidonicacid results in the production of proinflammatory mediators produced bythe action of enzymes such as COX-2. Inhibition of the swelling is adirect measure of the inhibition of the enzymes involved in thispathway. The results are set forth in FIG. 3, which shows the effects ofthree extracts delivered either orally by gavage or interperitoneally(IP) at two time points (24 hours and 1 hour). Free-B-Ring Flavonoidsisolated from S. baicaensis inhibited swelling when delivered by both IPand gavage although more efficacious by IP. (FIGS. 3A and B).Free-B-Ring Flavonoids isolated from S. orthocalyx inhibited thegeneration of these metabolites when given IP, but not orally, whereasextracts isolated from S. lateriflora, while being efficacious in vitro,had no effect in vivo (data not shown).

Air Pouch Model. Because Free-B-Ring Flavonoids isolated from S.baicaensis were the more efficacious in the ear swelling model, theywere also examined using the air pouch model of inflammation. Briefly,an air pouch was created on the back of the mice by injecting 3 mL ofsterile air. The air pouch was maintained by additional injections of 1mL of sterile air every other day for a period of one week. Animals weredosed using the same methods and concentrations described for theear-swelling model and injected with Zymosan (into the air pouch) toinitiate the inflammatory response. After four hours, the fluid withinthe pouch was collected and measured for the infiltration ofinflammatory cells, myeloperoxidase (MPO) activity (a measure ofcellular activation, degranulation), and tumor necrosis factor-α (TNF-α)production (a measure of activation). The results are set forth in FIG.4.

FIG. 4A shows the total number of cells collected from the air pouchfluid. While there was a strong response that was inhibited by controls(indomethacin), Free-B-Ring Flavonoids isolated from S. baicaensis didnot inhibit the infiltration of the inflammatory cells (chemotaxsis).Even though the chemotactic response was not diminished, the fluid wasexamined to determine whether the infiltrating cells have becomeactivated by measuring MPO activity and TNF-α production. FIGS. 4B and4C demonstrate that both MPO activity and TNFα production aresignificantly reduced when the extract is administered IP, but not bygavages. These data suggest that although the Free-B-Ring Flavonoids donot inhibit the chemotactic response induced by complement activationthey are still effective at reducing inflammation through the preventionof release and production of pro-inflammatory mediators.

Arachidonic Acid induced ear swelling. The ability of Free-B-RingFlavonoids to directly inhibit inflammation in vivo was measured aspreviously described (Greenspan et al. (1999) J. Med. Chem. 42:164-172;Young et al. (1984) J. Invest. Dermat. 82:367-371). Briefly, groups of 5Balb/C mice were given three dosages of test compounds as set forth inFIG. 4 either interperitoneally (I.P.) or orally by gavage, 24 hours and1 hour prior to the application of arachidonic acid (AA). AA in acetone(2 mg/15 μL) was applied to the left ear, and acetone (15 μL) as anegative control was applied to the right ear. After 1 hour the animalswere sacrificed by CO₂ inhalation and the thickness of the ears wasmeasured using an engineer's micrometer. Controls included animals givenAA, but not treated with anti-inflammatory agents, and animals treatedwith AA and indomethacin (I.P.) at 5 mg/kg.Air pouch model of inflammation. Air pouch models were adapted from themethods of Rioja et al. (2000) Eur. J. Pharm. 397:207-217. Air poucheswere established in groups of 5 Balb/C mice by injection of sterile air(3 mL) and maintained by additional injections of 1 mL every 2 days fora period of six days. Animals were given three dosages of test compoundsas shown in FIG. 4 either I.P. or orally by gavage, 24 hours and 1 hourprior to the injection of 1% Zymosan (1 mL) into the pouch. After 4hours, the animals were sacrificed by C0₂ inhalation and the air poucheswere lavaged with sterile saline (3 mL). The lavage fluid wascentrifuged and the total number of infiltrating cells determined.Supernatants were also retained and analyzed for myleoperoxidase (MPO)activity and the presence of TNF-α by ELISA as measures of activation.

Example 10 Development a Standardized Free-B-Ring Flavonoid Extract fromScutellaria Species

Scutellaria orthocalyx (500 mg of ground root) was extracted twice with25 mL of the following solvent systems. (1) 100% water, (2) 80:20water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5)20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, (8)60:40 methanol:THF. The extract solution was combined, concentrated anddried under low vacuum. Identification of chemical components wascarried out by High Pressure Liquid Chromatography using a PhotoDiodeArray detector (HPLC/PDA) and a 250 mm×4.6 mm C18 column. The chemicalcomponents were quantified based on retention time and PDA data usingBaicalein, Baicalin, Scutellarein, and Wogonin standards. The resultsare set forth in Table 7.

TABLE 7 Quantification of Free-B-Ring Flavonoids Extracted fromScutellaria orthocalyx Using Different Solvent Systems Total amount %Extraction Weight of % Extractible of Flavonoids Solvent Extract fromBioMass Flavonoids in Extract 100% water   96 mg 19.2% 0.02 mg 0.20%water:methanol 138.3 mg 27.7% 0.38 mg 0.38% (80:20) water:methanol 169.5mg 33.9% 0.78 mg 8.39% (60:40) water:methanol 142.2 mg 28.4% 1.14 mg11.26% (40:60) water:methanol 104.5 mg 20.9% 0.94 mg 7.99% (20:80) 100%methanol  57.5 mg 11.5% 0.99 mg 10.42% methanol:THF  59.6 mg 11.9% 0.89mg 8.76% (80:20) methanol:THF  58.8 mg 11.8% 1.10 mg 10.71% (60:40)

Scutellaria baicaensis (1000 mg of ground root) was extracted twiceusing 50 mL of a mixture of methanol and water as follows: (1) 100%water, (2) 70:30 water:methanol, (3) 50:50 water:methanol, (4) 30:70water:methanol, (5) 100% methanol. The extract solution was combined,concentrated and dried under low vacuum. Identification of the chemicalcomponents was carried out by HPLC using a PhotoDiode Array detector(HPLC/PDA), and a 250 mm×4.6 mm C18 column. The chemical components werequantified based on retention time and PDA data using Baicalein,Baicalin, Scutellarein, and Wogonin standards. The results are set forthin Table 8.

TABLE 8 Quantification of Free-B-Ring Flavonoids Extracted fromScutellaria baicaensis Using Different Solvent Systems Total amount %Extraction Weight of % Extractible of Flavonoids Solvent Extract fromBioMass Flavonoids in Extract 100% water 277.5 mg 27.8% 0.01 mg 0.09%water:methanol 338.6 mg 33.9% 1.19 mg 11.48% (70:30) water:methanol304.3 mg 30.4% 1.99 mg 18.93% (50:50) water:methanol 293.9 mg 29.4% 2.29mg 19.61% (30:70) 100% methanol 204.2 mg 20.4% 2.73 mg 24.51%

Example 11 Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoidson Pain Relieve of Rheumatoid Arthritis or Osteoarthritis of the Kneeand/or Hip

This clinical study was a single-center, randomized, double-blind,placebo-controlled study. Sixty subjects (n=60) with rheumatoidarthritis or osteoarthritis of the knee and/or hip were randomly placedinto one of the following four groups:

A₀ Placebo n = 15 Placebo A₁ Dose 1 n = 15 Univestin 250 mg/day (125 mgb.i.d.) A₂ Dose 2 n = 15 Univestin 500 mg/day (250 mg b.i.d.) A₃ ActiveControl n = 15 Celebrex 200 mg/day (100 mg b.i.d.)Univestin consists of a proprietary ratio of standardized extract ofScutellaria baicalensis Georgiwith a Baicalin content of 62.5% (w/w) andtotal Free-B-Ring Flavonoids>75% (w/w). Celebrex is a trade name for aprescription drug that is a COX-2 selective inhibitor.

The subjects were sex-matched and recruited from the ages of 40 to 75.Treatment consisted of oral administration for 60 days of the placebo oractive compound (Univestin or Celebrex) according to the above doseschedule. Subjects taking NSAIDs engaged in a two-week washout periodbefore the beginning of the study. Physical activity was not restricted.Subjects were free to withdraw from the trial at any time for anyreason. The efficacy of treatment was evaluated for 60 days after oraladministration by physicians using the Western Ontario and McMasterUniversities (WOMAC) Osteo-Arthritis Index. (See Lingard et al. (2001)The Journal of Bone & Joint Surgery 83(12):1856-1864; Soderman & Malchau(2000) Acta Orthop Scand. 71(1):39-46). This protocol was reviewed andapproved by an IRB board from the University of Montreal. Table 9 setsforth the WOMAC index scores before treatment (baseline scores) andTable 10 sets forth the changes in WOMAC index scores after treatmentfor 60 days. FIGS. 5 to 8 illustrate the results of the studygraphically.

TABLE 9 WOMAC Scores at Baseline before Treatment WOMAC SUBSCALE SCORESAT BASELINE UNIVESTIN UNIVESTIN WOMAC PLACEBO CELECOXIB 125 250 SUBSCALEMEAN SD MEAN SD MEAN SD MEAN SD PAIN 10.00 2.60 10.20 2.40 10.10 2.8010.30 2.50 STIFFNESS 4.80 1.00 4.70 1.30 4.90 1.50 4.70 1.20 PHYSICAL38.00 9.50 37.00 9.90 37.50 100.00 36.50 1.00 FUNTIONING WOMAC 52.8013.10 51.90 13.60 52.50 104.30 51.50 4.70 COMPOSITE SCORES Patient BMIin all groups ranged from 31 to 33 ± 6.5. No statistically significantdifference among treatment groups was observed.

TABLE 10 WOMAC scores after 60 days of treatments UNIVESTIN UNIVESTINPLACEBO CELECOXIB 250 mg 500 mg WOMAC % % % % SUBSCALE MEAN CHANGE MEANCHANGE MEAN CHANGE MEAN CHANGE PAIN −0.95 −9.50 −2.90 −28.43 −2.80−27.72 −2.90 −28.16 STIFFNESS −0.40 −8.33 −1.10 −23.40 −1.20 −24.49−1.40 −29.79 PHYSICAL −3.25 −8.55 −8.90 −24.05 −8.20 −21.87 −8.50 −23.29FUNTIONING WOMAC −4.60 −8.71 −12.90 −24.86 −12.20 −23.24 −12.80 −24.85COMPOSITE SCORES

1. A method for reducing joint dysfunction caused by peroxidase freeradical initiated joint wear and tear comprising administering to a hostin need thereof a therapeutically effective amount of a compositioncomprising a Free-B-Ring flavonoid extracted from Scutellaria or acomposition containing a mixture of Free-B-Ring flavonoids extractedfrom Scutellaria and a pharmaceutical acceptable carrier; wherein saidthe composition is administered at a dosage of 0.01 to 200 mg/kg of bodyweight of said host.
 2. The method of claim 1 wherein said Free-B-Ringflavonoid is selected from the group of compounds having the followingstructure:

wherein R₁, R₂, R₃ and R₄ are independently selected from the groupconsisting of —H, —OH, OR, a glycoside of a single or a combination ofmultiple sugars including, aldopentoses, methyl-aldopentoses,aldohexoses, ketohexoses, glucuronides and their chemical derivativesthereof; wherein R is an alkyl group having between 1-10 carbon atoms.3. The method of claim 1 wherein said Scutellaria is selected from thegroup consisting of Scutellaria baicalensis, Scutellaria lateriflora,Scutellaria radix and Scutellaria orthocalyx.
 4. The method of claim 1wherein said Free-B-Ring flavonoid is isolated from a plant part.
 5. Themethod of claim 4 wherein the Free-B-Ring flavonoid is isolated from aplant part selected from the group consisting of stems, stem barks,twigs, tubers, roots, root barks, young shoots, seeds, rhizomes,flowers, other reproductive organs, leaves and other aerial parts. 6.The method of claim 1 wherein the administration is selected from thegroup consisting of oral, topical, suppository, intravenous,intradermic, intragaster, intramuscular, intraperitoneal andintravenous.
 7. A method for reducing joint pain, immobility,discomfort, stiffness and inflexibility resulting from free radicalproduction by peroxidase comprising administering to a host in needthereof a therapeutically effective amount of a composition comprising aFree-B-Ring flavonoid extracted from Scutellaria or a compositioncontaining a mixture of Free-B-Ring flavonoids extracted fromScutellaria and a pharmaceutical acceptable carrier; wherein saidcomposition is administered at a dosage of 0.01 to 200 mg/kg of bodyweight of said host.
 8. The method of claim 7 wherein said Free-B-Ringflavonoid is selected from the group of compounds having the followingstructure:

wherein R₁, R₂, R₃ and R₄ are independently selected from the groupconsisting of —H, —OH, OR, a glycoside of a single or a combination ofmultiple sugars including, aldopentoses, methyl-aldopentoses,aldohexoses, ketohexoses, glucuronides and their chemical derivativesthereof; wherein R is an alkyl group having between 1-10 carbon atoms.9. The method of claim 7 wherein said Scutellaria is selected from thegroup consisting of Scutellaria baicalensis, Scutellaria lateriflora,Scutellaria radix and Scutellaria orthocalyx.
 10. The method of claim 7wherein said Free-B-Ring flavonoid is isolated from a plant part. 11.The method of claim 10 wherein the Free-B-Ring flavonoid is isolatedfrom a plant part selected from the group consisting of stems, stembarks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes,flowers, other reproductive organs, leaves and other aerial parts. 12.The method of claim 7 wherein the administration is selected from thegroup consisting of oral, topical, suppository, intravenous,intradermic, intragaster, intramuscular, intraperitoneal andintravenous.